Models of X-Linked Retinitis Pigmentosa

描述(申请人提供)：RPGTP酶调节基因(RPGR)的突变导致RP3型X连锁RP型视网膜色素变性，这是最常见和最严重的视网膜色素变性之一。这些突变中有一半以上发生在ORF15，这是一个功能未知的富含嘌呤的重复区域。在目前的资助中，我们精细定位了两个X连锁的犬视网膜退行性变(XLPRA1和2)，并在ORF15中发现了两个不同的致病微缺失。这些是ORF15相关疾病的唯一动物模型。由于这两种疾病的表型非常不同，它们之间的主要差异反映了RPGRORF15微缺失的性质。然而，在XLPRA1内部，尽管所有受影响的狗共享一个共同的稳定突变，源于单一创始人，但疾病严重程度存在显著差异。这显然不是环境因素或主要基因座的异质性造成的，而是作为独立于主要疾病基因的半显性性状分离而产生的。基于SNP的单倍型将被开发来测试已知与RPGR相互作用的基因，作为候选疾病修饰物。信息丰富的家系将被检查与疾病严重程度的共同隔离。如果没有发现这种关联，那么将进行基因组扫描，以确定具有潜在位置候选的基因组区域。 我们将利用在mRNA和蛋白质水平上的表达差异来识别与疾病相关的转录本和基因产物。核糖核酸酶保护试验将用于鉴定含有ORF15的转录本，并评估其在视网膜和非视网膜组织中的表达。因为这两个突变都改变了ORF15蛋白的长度和电荷，所以2D-Gel和多肽测序将被用来识别与疾病相关的亚型。将开发特定的抗体用于免疫化学研究，以评估每种异构体在发育和退化过程中的表达和定位。使用我们定制的狗视网膜基因芯片，我们将识别基因表达的变化，这些变化要么是突变特有的，要么是这两种疾病的共同特征。这将揭示视觉细胞疾病和退化背后的分子过程。此外，它将有助于指示何时发生可能限制治疗干预的不可逆转的代谢变化，并建立时间点，用于实施和评估我们计划使用AAV2/5假型载体的基因治疗。两个假说将通过基因治疗得到实验验证：a)停止突变(XLPRA1)引起的疾病是由需要基因替换的功能丧失引起的；以及b)移码突变(XLPRA2)引起的疾病是由有害的功能获得引起的，需要联合使用核酶敲除和基因替换进行早期干预。

DESCRIPTION (provided by applicant): Mutations in the RP GTPase regulator gene (RPGR) cause the RP3 form of X-linked RP, one of the most prevalent and severe forms of retinitis pigmentosa. Over half of these mutations occur in ORF15, a purine rich repetitive domain of unknown function. During the current grant we fine mapped two X-linked canine retinal degenerations (XLPRA1 and 2), and found 2 different causative rnicrodeletions in ORF15. These are the only animal models for ORF15-associated disease. Because the 2 disorders are phenotypically very distinct, the major differences between them reflect the nature of the RPGRORF15 microdeletions. Within XLPRA1 however, although all affected dogs share a common stable mutation originating from a single founder, significant variation in disease severity exists. This clearly results not from environmental factors or heterogeneity at the primary locus, but as a semi-dominant trait segregating independently of the primary disease locus. SNP-based haplotypes will be developed to test genes known to interact with RPGR, as candidate disease modifiers. Informative pedigrees will be examined for cosegregation with disease severity. If no such association is found, a genome scan will then be undertaken to define genomic regions with potential positional candidates. We will exploit differences in expression, at both the mRNA and protein level, to identify disease-associated transcripts and gene products. RNase protection assays will be used to identify ORF15 containing transcripts and evaluate expression in retinal vs. nonretinal tissues. Because both mutations alter the length and charge of the ORF15 proteins, 2D-gels and peptide sequencing will be used to identify disease relevant isoforms. Specific antibodies will be developed for immunochemical studies to evaluate expression and localization of each isoform during development and degeneration. Using our custom canine retinal cDNA microarray we will identify changes in gene expression that are either mutation specific or common to both disorders. This will reveal insights into the molecular processes underlying the disease and degeneration of visual cells. Further, it will help to indicate when irreversible metabolic changes occur that could limit therapeutic intervention, and establish time points for implementation and evaluation of our planned gene therapy using AAV 2/5 pseudotyped vectors. Two hypotheses will be tested experimentally with gene therapy: a) -that disease from the stop mutation (XLPRA1) is caused by loss of function requiring gene replacement; and b) -that disease from the frameshift mutation (XLPRA2) arises from a deleterious gain of function, necessitating early intervention with combined ribozyme knockdown and gene replacement.